The hedgehog genes encode inductive factors which regulative diverse aspects of vertebrate and invertebrate development. For example, Sonic hedgehog (Shh) is the key signal produced in the posterior limb but which organizes the anterior -posterior axis of the developing limb bud. Yet in spite of their importance, the mechanisms by which these proteins exert their effects is still poorly understood. The long term objective of this project is to elucidate the role Shh plays, with a focus on its function in limb development, and to understand the mechanism of its action, however whether this is a direct effect or accomplished via secondary signals remains controversial. We will directly address this issue by activating the hedgehog pathway cell autonomously, using an activated form of the downstream gene Smoothened; and by repressing the hedgehog pathway cell autonomously, using a dominant-negative form of the downstream transcription factor Gli, made by fusing an Engrailed repressor domain to the Gli DNA binding domain and assaying molecular targets. The signaling pathway by which the hedgehog genes act is only partially characterized, based on genetic analysis in Drosophila. We have recently isolated homologous of two genes in this pathway which were previously unknown in vertebrates: Fused (Fu) and Suppressor of Fused (Su(Fu)). We will study their expression, whether these proteins interact physically, and whether Fu and Su(Fu) will be downstream transcription factors encoded by the Gli genes. Genetic interactions between Fu and Su(Fu) will tested by viral misexpression and if necessary by making targeted mutations in mice. Two hybrid screens will be conducted with these proteins and with the receptor proteins Patched (Ptc) and Smoothened to try to fill in gaps in our understanding of the pathway. Finally we will address the distinct roles of different members of the hedgehog; directly comparing the activities of the three vertebrate hedgehog genes in limb duplication, left-right hedgehog; directly comparing the activities of the three vertebrate hedgehog genes in limb duplication, left-right hedgehog; directly comparing the activities of the three vertebrate hedgehog genes in limb duplication, left-right axis reversal, and bone differentiation assays; and misexpressing the different Gli and Ptc genes in the limb. In these experiments the Ptc and Gli genes, which are too large for the chick viral vector system, we will be misexpressed in limbs of transgenic mice.